Quantitation of insulin-like growth factor-I and insulin-like growth factor-II with high-resolution mass spectrometry

ABSTRACT

Methods are provided for determining the amount of an IGF-I and/or IGF-II protein in a sample using high resolution/high accuracy mass spectrometry. The methods generally comprise enriching an IGF-I and/or IGF-II protein in a sample, ionizing an IGF-I and/or IGF-II protein from the sample to generate IGF-I and/or IGF-II protein ions, and determining the amount of IGF-I and/or IGF-II protein ions with high resolution/high accuracy mass spectrometry.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. non-provisionalapplication Ser. No. 15/602,764, filed May 23, 2017, which iscontinuation of U.S. non-provisional application Ser. No. 12/939,996,filed Nov. 4, 2010, which claims priority to U.S. ProvisionalApplication Ser. No. 61/258,560, filed Nov. 5, 2009, and U.S.Provisional Application Ser. No. 61/408,535, filed Oct. 29, 2010, whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the quantitative analysis of large proteinsusing mass spectrometry.

BACKGROUND OF THE INVENTION

IGF-I is a hormone with a molecular structure similar to insulin. It isa peptide produced by the liver and contains 70 amino acids in a singlechain with three intramolecular disulfide bridges. IGF-I has a molecularweight of about 7,649 Da, and is highly protein bound in serum.Production is stimulated by growth hormone and can be retarded byundernutrition, growth hormone insensitivity, lack of growth hormonereceptors, or failure of a downstream signaling pathway. IGF-I plays animportant role in childhood growth and continues to have anaboliceffects in adults.

Insulin-like growth factor II (IGF-II) is also a hormone with amolecular structure similar to insulin. It is a single chain peptidethat contains 67 amino acids. IGF-II has a molecular weight of about7,505 Da, and is highly protein bound in serum. IGF-II is used as anadjunct to insulin-like growth factor I (IGF-I) in clinical evaluationof growth hormone-related disorders. IGF-II plays a role primarily infetal growth and development by interplaying with IGF-I and differentcell surface receptors and circulating binding proteins to modulatetissue growth. IGF-II levels are reduced in children and adults as aresult of growth hormone deficiency or malnutrition. Increased IGF-IIserum levels may be observed in acromegaly or with exogenousadministration of IGF-I. Thus, measurement of circulating IGF-II levels(i.e. in plasma/serum) is an important tool in management of severalgrowth hormone-related disorders. In addition, measurement ofcirculating IGF-II is also a valuable tool in various epidemiologicalresearch areas and clinical trials.

IGF-I and IGF-II have proven to be particularly challenging toquantitatively analyze with a “bottom up” approach (i.e., enzymaticdigestion and quantitation of one or more of the resulting peptides).For example, de Kock, et al. reported that typical trypsin,chymotrypsin, and pepsin digestion methods result in low digestion yieldand non-specific enzyme cleavage. de Kock, et al., Rapid Commun. MassSpectrom., 2001, 15:1191-97. de Kock, et al. suggested that theunsatisfactory digestion results were likely due to steric restrictionof the proteolytic enzymes by IGF-I's three disulfide bonds. Id.

Efforts have been made to develop methods to analyze IGF-I and IGF-II,including by a variety of mass spectrometric techniques. For example,analysis of IGF-I has been reported using LC-ion trap MS with single ionmonitoring (SIM). See, e.g., Id.; Bobin, et al., Analyst, 2001,126:1996-2001; and Popot, et al., Chromatographia, 2001, 54:737-741(Popot I). These references disclose LC-ESI-ion trap MS of intact IGF-Iat charge states ranging from 4+ to 9+ in SIM mode. More recently,LC-MS/MS techniques using multiple reaction monitoring (MRM) have beendisclosed. Popot II reports quantitative analysis of IGF-I at chargestates ranging from 4+ to 9+ using LC-ion trap MS with SIM, followed byqualitative confirmation of the analyte with LC-ion trap MS/MS usingMRM. Popot, et al., Anal Bioanal Chem, 2008, 390:1843-52 (Popot II). TheMRM experiments use selected multicharged IGF-I ions (7+ and 8+) asprecursor ions in the fragmentation experiments. Bredehoft, et al.reports using an orbitrap mass spectrometric instrument to qualitativelydetermine the mass to charge ratios of precursor and product ions ofIGF-I and use of the identified ions for subsequent IGF-I quantitationusing LC-triple quadrupole MS/MS. Bredehoft, et al., Rapid Commun. MassSpectrom., 2008, 22:477-485.

Similarly, analysis of IGF-II has been reported using enzymaticdigestion followed by mass spectrometric analysis of the digestionproducts. See, e.g., Smith et al., J. Biol. Chem., 1989, 16:9314-9321;and Bayne et al., Peptide Research, 1990, 6:271-273. Other studies havebeen reported for analysis of mass spectrometric detection of intactIGF-II. See, e.g., Hampton et al., J. Biol. Chem., 1989, 32:19155-19160;Hayne, et al., J. Chromatog. 1991, 52:391-402; Wadensten, et al.,Biotech. and App. Biochem., 1991, 13:412-421; Jespersen, et al., J. MassSpectrom., 1996, 31:893-900; and Nelson, et al., J. Proteome, 2004,3:851-855. These references report detection of intact IGF-II withplasma desorption and MALDI-TOF mass spectrometric techniques. Hamptonet al. detects charge states ranging from 1+ to 3+. All but Hampton etal. detect IGF-II at a 1+ charge state.

SUMMARY OF THE INVENTION

The methods described herein are for mass spectrometric determination ofthe amount of an insulin-like growth factor I (IGF-I) protein and/or aninsulin-like growth factor II (IGF-II) protein in a sample with highresolution/high accuracy mass spectrometry. The methods have simplifiedpre-analytic steps which only optionally include protein fragmentation,digestion, and/or detection of protein fragments. Preferably, nofragmentation or digestion of the intact protein or proteins isconducted following sample collection.

In a first aspect of the present invention, the amount of an IGF-Iprotein or fragment thereof in a sample is determined by massspectrometry methods which include subjecting an IGF-I protein orfragment thereof from the sample to ionization under conditions suitableto produce one or more IGF-I ions detectable by mass spectrometry; anddetermining the amount of one or more IGF-I ions by high resolution/highaccuracy mass spectrometry. The amount of the determined IGF-I ion orions is related to the amount of the IGF-I protein or fragment thereofin the sample. In some embodiments, the IGF-I protein or fragmentthereof is native to the sample. In some embodiments, the IGF-I proteinor fragment thereof is intact long R3 IGF-I or a fragment thereof. Insome embodiments, the IGF-I protein is intact long R3 IGF-I.

In a second aspect, the amount of an IGF-II protein or fragment thereofin a sample is determined by mass spectrometry methods which includesubjecting the IGF-II protein or fragment thereof in the sample toionization under conditions suitable to produce one or more IGF-II ionsdetectable by mass spectrometry; and determining the amount of one ormore IGF-II ions by high resolution/high accuracy mass spectrometry. Theamount of the determined IGF-II ion or ions is related to the amount ofthe IGF-II protein or fragment thereof in the sample. Preferably, theIGF-II protein is native to the sample and intact.

In a third aspect, the amounts of both an IGF-I protein or fragmentthereof and an IGF-II protein or fragment thereof are simultaneouslydetermined by mass spectrometry methods which include subjecting anIGF-I protein or fragment thereof and an IGF-II protein or fragmentthereof from the sample to ionization under conditions suitable toproduce one or more IGF-I ions and one or more IGF-II ions detectable bymass spectrometry; and determining the amount of one or more IGF-I ionsand one or more IGF-II ions by high resolution/high accuracy massspectrometry. The amount of the determined IGF-I and IGF-II ions arerelated to the amount of the IGF-I and IGF-II proteins or fragmentsthereof in the sample. Preferably, the IGF-I and IGF-II proteins arenative to the sample and intact.

In some embodiments, the sample may be purified by solid phaseextraction (SPE) prior to ionization. In some embodiments, the samplemay be purified by high performance liquid chromatography (HPLC) priorto ionization. In related embodiments, the sample may be purified withboth SPE and HPLC prior to ionization, and the purification mayoptionally be conducted with on-line processing.

In some embodiments, the high resolution/high accuracy mass spectrometryis conducted with a resolving power (FWHM) of greater than or equal toabout 10,000, such as greater than or equal to about 15,000, such asgreater than or equal to about 20,000, such as greater than or equal toabout 25,000. In some embodiments, the high resolution/high accuracymass spectrometry is conducted at an accuracy of less than or equal toabout 50 ppm, such as less than or equal to about 20 ppm, such as lessthan or equal to about 10 ppm, such as less than or equal to about 5ppm; such as less than or equal to about 3 ppm. In some embodiments,high resolution/high accuracy mass spectrometry is conducted at aresolving power (FWHM) of greater than or equal to about 10,000 and anaccuracy of less than or equal to about 50 ppm. In some embodiments, theresolving power is greater than about 15,000 and the accuracy is lessthan or equal to about 20 ppm. In some embodiments, the resolving poweris greater than or equal to about 20,000 and the accuracy is less thanor equal to about 10 ppm; preferably resolving power is greater than orequal to about 25,000 and accuracy is less than or equal to about 5 ppm,such as less than or equal to about 3 ppm.

In some embodiments, the high resolution/high accuracy mass spectrometrymay be conducted with an orbitrap mass spectrometer, a time of flight(TOF) mass spectrometer, or a Fourier transform ion cyclotron resonancemass spectrometer (sometimes known as a Fourier transform massspectrometer). In some embodiments, the sample may include a biologicalsample; preferably plasma or serum.

In some embodiments, the one or more IGF-I ions detectable by massspectrometry are one or more ions selected from the group consisting ofions with m/z within the ranges of about 850.8±2, 957.1±2, 1093.7±2, and1275.8±2. Ions within these ranges correspond to IGF-I ions with chargesof 9+, 8+, 7+, and 6+, respectively, and predominantly fall within theranges of the cited m/z values±1. Preferably the one or more IGF-I ionscomprise one or more ions selected from the group consisting of IGF-Iions with m/z within the ranges of 957.1±2 and 1093.7±2. IGF-I ionswithin the range of 1093.7±2 preferably comprise one or more IGF-I ionsselected from the group consisting of IGF-I ions with m/z of about1091.94±0.1, 1092.80±0.1, 1092.94±0.1, 1093.09±0.1, 1093.23±0.1,1093.37±0.1, 1093.52±0.1, 1093.66±0.1, 1093.80±0.1, 1093.95±0.1,1094.09±0.1, 1094.23±0.1, 1094.38±0.1, 1094.52±0.1, 1094.66±0.1, and1095.37±0.1. In some embodiments, relating the amount of one or moreIGF-I ions detected by mass spectrometry to the amount of an IGF-Iprotein in the sample includes comparison to an internal standard; suchas a human or non-human IGF-I protein (e.g., intact recombinant mouserecombinant mouse IGF-I). The internal standard may optionally beisotopically labeled.

In some embodiments, the one or more IGF-II ions detectable by massspectrometry are one or more IGF-II ions selected from the groupconsisting of IGF-II ions with m/z within the ranges of about 934.69±2,1068.07±2, 1245.92±2, and 1494.89±2. Ions within these ranges correspondto IGF-II ions with charges of 8+, 7+, 6+, and 5+, respectively, andpredominantly fall within the ranges of the cited m/z values±1.Preferably the one or more IGF-II ions comprise an IGF-II ion selectedfrom the group consisting of IGF-II ions with m/z of about 1067.36±0.1,1067.51±0.1, 1067.65±0.1, 1067.80±0.1, 1067.94±0.1, 1068.08±0.1,1068.23±0.1, 1068.37±0.1, 1068.51±0.1, 1068.65±0.1, 1068.80±0.1,1068.94±0.1, and 1069.08±0.1; preferably, the one or more IGF-II ionsare selected from the group consisting of ions with m/z of about1067.94±0.1 and 1068.08±0.1. In some embodiments, relating the amount ofone or more IGF-II ions detected by mass spectrometry to the amount ofIGF-II protein in the sample includes comparison to an internalstandard; such as a human or non-human IGF-II protein (e.g., intactrecombinant mouse IGF-II).

In some embodiments, the amounts of native intact IGF-I and/or IGF-II ina sample are determined by mass spectrometry methods which includesubjecting intact IGF-I and/or IGF-II, native to the sample and purifiedby solid phase extraction (SPE) and high performance liquidchromatography (HPLC), to ionization under conditions suitable toproduce one or more IGF-I and/or IGF-II ions detectable by massspectrometry. If the amount of native intact IGF-I is determined, theone or more IGF-I ions include one or more IGF-I ions selected from thegroup consisting of IGF-I ions with m/z within the ranges of about850.8±2, 957.1±2, 1093.7±2, and 1275.8±2. If the amount of native intactIGF-II is determined , the one or more IGF-II ions include one or moreIGF-II ions selected from the group consisting of IGF-II ions with m/zwithin the ranges of about 934.69±2, 1068.07±2, 1245.92±2, and1494.89±2. Again, for both IGF-I and IGF-II, ions within the aboveranges fall predominantly within the ranges of the indicted m/z value±1.The amount of the one or more IGF-I and/or IGF-II ions are thendetermined by high resolution/high accuracy mass spectrometry; whereinthe amount of the one or more IGF-I and/or IGF-II ions is used todetermine the amount of native intact IGF-I and/or IGF-II in the sample.In these embodiments, the high resolution/high accuracy massspectrometry is conducted with an orbitrap or TOF mass spectrometer, andSPE and HPLC may be conducted in an on-line fashion.

The features of the embodiments listed above may be combined withoutlimitation for use in methods of the present invention.

In certain preferred embodiments, mass spectrometry is performed inpositive ion mode. Alternatively, mass spectrometry is performed innegative ion mode. The preferred ionization technique used in methodsdescribed herein is electrospray ionization (ESI). Electrosprayionization may be conducted, for example, with a heated ionizationsource.

In preferred embodiments, one or more separately detectable internalstandards is provided in the sample, the amount of which is alsodetermined in the sample. In these embodiments, all or a portion of boththe analyte of interest and the one or more internal standards isionized to produce a plurality of ions detectable in a massspectrometer, and one or more ions produced from each are detected bymass spectrometry. The internal standards may be selected from the groupconsisting of intact non-human IGF-I (e.g., isotopically labeled orunlabeled intact recombinant mouse IGF-I), an isotopically labeledintact human IGF-I protein, an intact non-human IGF-II protein(e.g.,isotopically labeled or unlabeled intact recombinant mouse IGF-II), andan isotopically labeled intact human IGF-II protein.

In other embodiments, the amount of an intact IGF-I and/or IGF-IIprotein in a sample may be determined by comparison to one or moreexternal reference standards. Exemplary external reference standardsinclude blank plasma or serum spiked with an isotopically labeled orunlabeled, intact human or non-human IGF-I and/or IGF-II protein (e.g.,isotopically labeled or unlabeled intact recombinant mouse IGF-I and orIGF-II).

In some embodiments, an isotopic signature comprising mass spectrometricpeaks from two or more molecular isotopic forms of an analyte may beused to confirm the identity of an analyte being studied. In otherembodiments, a mass spectrometric peak from one or more isotopic formsmay be used to quantitate the analyte of interest. In some relatedembodiments, a single the mass spectrometric peak from one isotopic formmay be used to quantitate an analyte of interest. In other relatedembodiments, a plurality of isotopic peaks may be used to quantitate ananalyte. The plurality of peaks may be subject to any appropriatemathematical treatment. Several mathematical treatments are known in theart and include, but are not limited to, summing the area under multiplepeaks, or averaging the response from multiple peaks.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “IGF-I protein” refers to full-length IGF-Ipolypeptides or fragments thereof, as well as full-length IGF-I variantpolypeptides or fragments thereof. IGF-I variants include, for example,long R3 IGF-I, which is an 83 amino acid analog of IGF-I comprising thecomplete human IGF-I sequence with the substitution of an Arg(R) for theGlu(E) at position three (hence R3) and a 13 amino acid extensionpeptide at the N terminus. This analog of IGF-I has been produced withthe purpose of increasing the biological activity of the IGF-I peptide.The mass of this analog is about 9111.4 Daltons, thus multiply chargedlong R3 IGF-I ions may be observed with m/z ratios of about 1014.1±1,1140.7±1, 1303.5±1, and 1520.6±1. Other IGF-I variant polypeptides arereadily recognized by one of skill in the art, including for examplefull-length IGF-I polypeptides or fragments thereof that have beenchemically modified. Exemplary chemical modifications may includereduction of one or more disulfide bridges or alkylation of one or morecystines. These exemplary chemical modifications result in an increasein the mass of an IGF-I variant polypeptide relative to the mass of thecorresponding unmodified IGF-I polypeptide. Reduction of one or moredisulfide bridges results in a relatively minor change in the mass ofthe molecule, with the resulting mass to charge ratios falling withinthe mass to charge ratio ranges described herein. Other chemicalmodifications that result in a mass deviation from an unmodified IGF-Ipolypeptide are also encompassed within the meaning of IGF-I protein.One skilled in the art understands that the addition of atoms to anIGF-I protein by chemical modification will result in an observedincrease in the mass to charge ratios during mass spectrometry. Thus,IGF-I protein variants that result from chemical modification areincluded within the meaning IGF-I protein and detectable in accordancewith the methods of the invention.

As used herein, the term “IGF-II protein” refers to full-length IGF-IIpolypeptides or fragments thereof, as well as full-length IGF-II variantpolypeptides or fragments thereof. IGF-II variant polypeptides arereadily recognized by one of skill in the art, including for examplefull-length IGF-II polypeptides or fragments thereof that have beenchemically modified. Exemplary chemical modifications may includereduction of one or more disulfide bridges or alkylation of one or morecystines. These exemplary chemical modifications result in an increasein the mass of an IGF-II variant polypeptide relative to the mass of thecorresponding unmodified IGF-II polypeptide. Reduction of one or moredisulfide bridges results in a relatively minor change in the mass ofthe molecule, with the resulting m/z falling within the m/z rangesdescribed herein. Other chemical modifications that result in a massdeviation from an unmodified IGF-II polypeptide are also encompassedwithin the meaning of IGF-II protein. One skilled in the art understandsthat the addition of atoms to an IGF-II protein by chemical modificationwill result in an observed increase in the mass to charge ratios duringmass spectrometry. Thus, IGF-II protein variants that result fromchemical modification are included within the meaning IGF-II protein anddetectable in accordance with the methods of the invention.

As used here, the term “intact” as describing a polypeptide refers tothe full-length (i.e., unfragmented) polypeptide. Intact IGF-I, forexample, is a polypeptide containing 70 amino acid residues, and intactlong R3 IGF-I is an 83 amino acid analog of IGF-I comprising thecomplete human IGF-I sequence with the substitution of an Arg(R) for theGLu(E) at position three (hence R3) and a 13 amino acid extensionpeptide at the N terminus. Non-intact forms of IGF-I and/or IGF-IIproteins (i.e., fragments) may also be detected by the methods describedherein. For example, fragments of IGF-I and/or IGF-II proteins with amolecular weight of about 1,000 Daltons or larger, such as about 1500Daltons or larger, such as about 2000 Daltons or larger, such as about2500 Daltons or larger, such as about 3000 Daltons or larger, such asabout 4000 Daltons or larger, such as about 5000 Daltons or larger, suchas about 6000 Daltons or larger, such as about 7000 Daltons or largermay be detected by methods described herein.

The term “purification” or “purifying” refers to a procedure thatenriches the amount of one or more analytes of interest relative toother components in the sample that may interfere with detection of theanalyte of interest. Although not required, “purification” maycompletely remove all interfering components, or even all material otherthan the analyte of interest. Purification of the sample by variousmeans may allow relative reduction of one or more interferingsubstances, e.g., one or more substances that may or may not interferewith the detection of selected parent or daughter ions by massspectrometry. Relative reduction as this term is used does not requirethat any substance, present with the analyte of interest in the materialto be purified, is entirely removed by purification.

The term “sample” refers to any sample that may contain an analyte ofinterest. As used herein, the term “body fluid” means any fluid that canbe isolated from the body of an individual. For example, “body fluid”may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like. In preferred embodiments, the samplecomprises a body fluid sample; preferably plasma or serum.

The term “solid phase extraction” or “SPE” refers to a process in whicha chemical mixture is separated into components as a result of theaffinity of components dissolved or suspended in a solution (i.e.,mobile phase) for a solid through or around which the solution is passed(i.e., solid phase). In some instances, as the mobile phase passesthrough or around the solid phase, undesired components of the mobilephase may be retained by the solid phase resulting in a purification ofthe analyte in the mobile phase. In other instances, the analyte may beretained by the solid phase, allowing undesired components of the mobilephase to pass through or around the solid phase. In these instances, asecond mobile phase is then used to elute the retained analyte off ofthe solid phase for further processing or analysis. SPE may operate viaa unitary or mixed mode mechanism. As used herein, SPE can be conductedwith an extraction column or cartridge such as, for example, a turbulentflow liquid chromatography (TFLC) column. Mixed mode mechanisms utilizeion exchange and hydrophobic retention in the same column; for example,the solid phase of a mixed-mode SPE column may exhibit strong anionexchange and hydrophobic retention; or may exhibit strong cationexchange and hydrophobic retention.

The term “chromatography” refers to a process in which a chemicalmixture carried by a liquid or gas is separated into components as aresult of differential distribution of the chemical entities as theyflow through a stationary solid phase.

The term “liquid chromatography” or “LC” means a process of selectiveretardation of one or more components of a fluid solution as the fluiduniformly percolates through a column of a finely divided substance, orthrough capillary passageways. The retardation results from thedistribution of the components of the mixture between one or morestationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of separationtechniques which employ “liquid chromatography” include reverse phaseliquid chromatography (RPLC), high performance liquid chromatography(HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes knownas high turbulence liquid chromatography (HTLC) or high throughputliquid chromatography). In some embodiments, an SPE column may be usedin combination with an LC column. For example, a sample may be purifiedwith a TFLC extraction column, followed by additional purification witha HPLC analytical column.

The term “high performance liquid chromatography” or “HPLC” (sometimesknown as “high pressure liquid chromatography”) refers to liquidchromatography in which the degree of separation is increased by forcingthe mobile phase under pressure through a stationary phase, typically adensely packed column.

The term “turbulent flow liquid chromatography” or “TFLC” (sometimesknown as high turbulence liquid chromatography or high throughput liquidchromatography) refers to a form of chromatography that utilizesturbulent flow of the material being assayed through the column packingas the basis for performing the separation. TFLC has been applied in thepreparation of samples containing two unnamed drugs prior to analysis bymass spectrometry. See, e.g., Zimmer et al., J Chromatogr A 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain TFLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar. In laminar flow, the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

The term “gas chromatography” or “GC” refers to chromatography in whichthe sample mixture is vaporized and injected into a stream of carriergas (as nitrogen or helium) moving through a column containing astationary phase composed of a liquid or a particulate solid and isseparated into its component compounds according to the affinity of thecompounds for the stationary phase.

The terms “on-line” and “inline”, for example as used in “on-lineautomated fashion” or “on-line extraction” refers to a procedureperformed without the need for operator intervention. In contrast, theterm “off-line” as used herein refers to a procedure requiring manualintervention of an operator. Thus, if samples are subjected toprecipitation, and the supernatants are then manually loaded into anautosampler, the precipitation and loading steps are off-line from thesubsequent steps. In various embodiments of the methods, one or moresteps may be performed in an on-line automated fashion.

The term “mass spectrometry” or “MS” refers to an analytical techniqueto identify compounds by their mass. MS refers to methods of filtering,detecting, and measuring ions based on their mass-to-charge ratio, or“m/z”. MS technology generally includes (1) ionizing the compounds toform charged compounds; and (2) detecting the molecular weight of thecharged compounds and calculating a mass-to-charge ratio. The compoundsmay be ionized and detected by any suitable means. A “mass spectrometer”generally includes an ionizer and an ion detector. In general, one ormore molecules of interest are ionized, and the ions are subsequentlyintroduced into a mass spectrometric instrument where, due to acombination of magnetic and electric fields, the ions follow a path inspace that is dependent upon mass (“m”) and charge (“z”). See, e.g.,U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;”U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem MassSpectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics BasedOn Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis2000, 21: 1164-67.

As used herein, “high resolution/high accuracy mass spectrometry” refersto mass spectrometry conducted with a mass analyzer capable of measuringthe mass to charge ratio of a charged species with sufficient precisionand accuracy to confirm a unique chemical ion. Confirmation of a uniquechemical ion is possible for an ion when individual isotopic peaks fromthat ion are readily discernable. The particular resolving power andmass accuracy necessary to confirm a unique chemical ion varies with themass and charge state of the ion.

As used herein, the term “resolving power” or “resolving power (FWHM)”(also known in the art as “m/Δm_(50%)”) refers to an observed mass tocharge ratio divided by the width of the mass peak at 50% maximum height(Full Width Half Maximum, “FWHM”). The effect of differences inresolving power is illustrated in FIGS. 1A-C, which show theoreticalmass spectra of an ion with a m/z of about 1093. FIG. 1A shows atheoretical mass spectrum from a mass analyzer with resolving power ofabout 3000 (a typical operating condition for a conventional quadrupolemass analyzer). As seen in FIG. 1A, no individual isotopic peaks arediscernable. By comparison, FIG. 1B shows a theoretical mass spectrumfrom a mass analyzer with resolving power of about 10,000, with clearlydiscernable individual isotopic peaks. FIG. 1C shows a theoretical massspectrum from a mass analyzer with resolving power of about 12,000. Atthis highest resolving power, the individual isotopic peaks contain lessthan 1% contribution from baseline.

As used herein a “unique chemical ion” with respect to mass spectrometryrefers a single ion with a single atomic makeup. The single ion may besingly or multiply charged.

As used herein, the term “accuracy” (or “mass accuracy”) with respect tomass spectrometry refers to potential deviation of the instrumentresponse from the true m/z of the ion investigated. Accuracy istypically expressed in parts per million (ppm). The effect ofdifferences in mass accuracy is illustrated in FIGS. 2A-D, which showthe boundaries of potential differences between a detected m/z and theactual m/z for a theoretical peak at m/z of 1093.52094. FIG. 2A showsthe potential range of detected m/z at an accuracy of 120 ppm. Bycontrast, FIG. 2B shows the potential range of detected m/z at anaccuracy of 50 ppm. FIGS. 2C and 2D show the even narrower potentialranges of detected m/z at accuracies of 20 ppm and 10 ppm.

High resolution/high accuracy mass spectrometry methods of the presentinvention may be conducted on instruments capable of performing massanalysis with FWHM of greater than 10,000, 15,000, 20,000, 25,000,50,000, 100,000, or even more. Likewise, methods of the presentinvention may be conducted on instruments capable of performing massanalysis with accuracy of less than 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5ppm, 3 ppm, or even less. Instruments capable of these performancecharacteristics may incorporate certain orbitrap mass analyzers,time-of-flight (“TOF”) mass analyzers, or Fourier-transform ioncyclotron resonance mass analyzers. In preferred embodiments, themethods are carried out with an instrument which includes an orbitrapmass analyzer or a TOF mass analyzer.

The term “orbitrap” describes an ion trap consisting of an outerbarrel-like electrode and a coaxial inner electrode. Ions are injectedtangentially into the electric field between the electrodes and trappedbecause electrostatic interactions between the ions and electrodes arebalanced by centrifugal forces as the ions orbit the coaxial innerelectrode. As an ion orbits the coaxial inner electrode, the orbitalpath of a trapped ion oscillates along the axis of the central electrodeat a harmonic frequency relative to the mass to charge ratio of the ion.Detection of the orbital oscillation frequency allows the orbitrap to beused as a mass analyzer with high accuracy (as low as 1-2 ppm) and highresolving power (FWHM) (up to about 200,000). A mass analyzer based onan orbitrap is described in detail in U.S. Pat. No. 6,995,364,incorporated by reference herein in its entirety. Use of orbitrapanalyzers has been reported for qualitative and quantitative analyses ofvarious analytes. See, e.g., U.S. Patent Application Pub. No.2008/0118932 (filed Nov. 9, 2007); Bredehoft, et al., Rapid Commun. MassSpectrom., 2008, 22:477-485; Le Breton, et al., Rapid Commun. MassSpectrom., 2008, 22:3130-36; Thevis, et al., Mass Spectrom. Reviews,2008, 27:35-50; Thomas, et al., J. Mass Spectrom., 2008, 43:908-15;Schenk, et al., BMC Medical Genomics, 2008, 1:41; and Olsen, et al.,Nature Methods, 2007, 4:709-12.

The term “operating in negative ion mode” refers to those massspectrometry methods where negative ions are generated and detected. Theterm “operating in positive ion mode” as used herein, refers to thosemass spectrometry methods where positive ions are generated anddetected.

The term “ionization” or “ionizing” refers to the process of generatingan ion having a net electrical charge equal to one or more electronunits. Negative ions are those having a net negative charge of one ormore electron units, while positive ions are those having a net positivecharge of one or more electron units.

The term “selective ion monitoring” is a detection mode for a massspectrometric instrument in which only ions within a relatively narrowmass range, typically about one mass unit or less, are detected.

“Multiple reaction mode,” sometimes known as “selected reactionmonitoring,” is a detection mode for a mass spectrometric instrument inwhich a precursor ion and one or more fragment ions are selectivelydetected.

The terms “lower limit of quantification”, “lower limit of quantitation”or “LLOQ” refer to the point where measurements become quantitativelymeaningful. The analyte response at this LLOQ is identifiable, discreteand reproducible with a relative standard deviation (RSD %) of less than20% and an accuracy of 85% to 115%.

The term “limit of detection” or “LOD” is the point at which themeasured value is larger than the uncertainty associated with itsmeasurement. The LOD is defined as four times the RSD of the mean at thezero concentration.

The term “simultaneous” as applied to simultaneously detecting theamount of two or more analytes from a sample means acquiring datareflective of the amount of the two or more analytes in the sample fromthe same sample injection. The data for each analyte may be acquiredsequentially or in parallel, depending on the instrumental techniquesemployed. For example, a single sample containing two analytes, such asintact IGF-I and IGF-II proteins, may be injected into a HPLC column,which may then elute each analyte one after the other, resulting inintroduction of the analytes into a mass spectrometer sequentially.Determining the amount of each of these two analytes is simultaneous forthe purposes herein, as both analytes result from the same sampleinjection into the HPLC.

An “amount” of an analyte in a body fluid sample refers generally to anabsolute value reflecting the mass of the analyte detectable in volumeof sample. However, an amount also contemplates a relative amount incomparison to another analyte amount. For example, an amount of ananalyte in a sample can be an amount which is greater than a control ornormal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show theoretical mass spectra of an ion with a m/z of about1093 as analyzed by a mass analyzer with resolving power of about 3000(FIG. 1A), about 10,000 (FIG. 1B), and about 12,000 (FIG. 1C).

FIGS. 2A-D show potential deviation of instrument response from the truem/z of the ion investigated for a theoretical peak at m/z of 1093.52094at a mass accuracy of 120 ppm (FIG. 2A), a mass accuracy of 50 ppm (FIG.2B), a mass accuracy of 20 ppm (FIG. 2C), and at a mass accuracy of 10ppm (FIG. 2D).

FIG. 3 shows an exemplary spectrum across a m/z range of about 800 to1300 for intact IGF-I generated with an orbitrap mass spectrometer.Details are discussed in Example 2.

FIG. 4 shows an exemplary spectrum across a m/z range of about 1091 to1096 for intact IGF-I generated with an orbitrap mass spectrometer.Details are discussed in Example 2.

FIG. 5 shows a plot of analytical results for various on-columnquantities of intact IGF-I. Details are discussed in Example 2.

FIG. 6A shows an exemplary spectrum for about 40 fmol (on-column) intactIGF-I across a m/z range of about 1090 to 1098. FIG. 6B shows thecorresponding extracted ion chromatogram (EIC). Details are discussed inExample 3.

FIG. 7A shows an exemplary spectrum for about 650 fmol (on-column)intact IGF-I across a m/z range of about 1090 to 1098. FIG. 7B shows thecorresponding extracted ion chromatogram (EIC). Details are discussed inExample 3.

FIGS. 8A and 8B show the observed and calculated spectra of intact IGF-Iacross the m/z range of about 1092 to 1095.3. Details are discussed inExample 3.

FIGS. 9A-B show plots of analytical results for various on columnquantities of intact IGF-I. The full concentration range tested isdepicted in FIG. 9A, with an expanded view of the low end of theconcentration range depicted in FIG. 9B. Details are discussed inExample 3.

FIG. 10 shows a plot of the linearity of response for intact IGF-I atconcentrations ranging from 15 ng/mL to 2000 ng/mL. Details arediscussed in Example 4.

FIG. 11 shows an exemplary spectrum across a m/z range of about 500 to1900 for intact IGF-II generated with a high resolution/high accuracyTOF mass spectrometer. Details are discussed in Example 11.

FIG. 12 shows an exemplary spectrum across a m/z range of about 1066 to1070 for intact IGF-II generated with an high resolution/high accuracyTOF mass spectrometer. Details are discussed in Example 11.

FIG. 13 shows a plot of the linearity of response for intact IGF-II atconcentrations ranging from 15 ng/mL to 2000 ng/mL. Details arediscussed in Example 11.

FIG. 14 shows comparison of analytical results for quantitation ofintact IGF-II by the instant LC-MS methods versus analysis by IRMA.Details are discussed in Example 15.

FIGS. 15A, B, and C show an exemplary Total Ion Chromatogram (TIC) andExtracted Ion Chromatograms (EIC) from simultaneous quantitation ofIGF-II and IGF-I. FIG. 15A shows the TIC, FIG. 15B shows the EIC forIGF-II, and FIG. 15C shows the EIC for IGF-I. Details are discussed inExample 18.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring the amount of an IGF-I and/or IGF-IIprotein. More specifically, high accuracy/high resolution massspectrometric methods are described for ionizing intact IGF-I and/orintact IGF-II, or fragments thereof, and detecting ions producedthereby. These methods may include purifying intact IGF-I and/or intactIGF-II, or fragments thereof, in the sample prior to ionization and massspectrometry. However, the methods may be performed without purifyingthe sample with chromatography. Preferred embodiments are particularlywell suited for application in large clinical laboratories for automatedintact IGF-I and/or intact IGF-II, or fragment, quantification.Additionally, certain embodiments presented herein provide methods forIGF-I and/or IGF-II quantitation that are insensitive to interferencefrom binding proteins that may also be present in the sample, such as,for example, IGFBP-3.

While the examples discussed below demonstrate quantitation of intacthuman IGF-I and/or IGF-II, other IGF-I and/or IGF-II proteins may alsobe analyzed by the methods described herein. For example, intactnon-human IGF-I and/or IGF-II (e.g., isotopically labeled or unlabeledintact recombinant mouse IGF-I and/or IGF-II), isotopically labeledintact human IGF-I and/or IGF-II, or long R3 IGF-I, or fragmentsthereof, in suitable test samples may all be quantitated with thefollowing methods.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma and serum. Such samples may beobtained, for example, from a patient; that is, a living person, male orfemale, presenting oneself in a clinical setting for diagnosis,prognosis, or treatment of a disease or condition.

The present invention also contemplates kits for an IGF-I and/or IGF-IIprotein quantitation assay. A kit for an IGF-I and/or IGF-II proteinquantitation assay may include a kit comprising the compositionsprovided herein. For example, a kit may include packaging material andmeasured amounts of an isotopically labeled internal standard in amountssufficient for at least one assay. Typically, the kits will also includeinstructions recorded in a tangible form (e.g., contained on paper or anelectronic medium) for using the packaged reagents for use in an IGF-Iand/or IGF-II protein quantitation assay.

Quality control (QC) pools having known concentrations, for use inembodiments of the present invention, are preferably prepared using amatrix similar to the intended sample matrix.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, an IGF-I protein may beenriched relative to one or more other components in the sample (e.g.other proteins) by various methods known in the art, including forexample, solid phase extraction (SPE), LC, filtration, centrifugation,thin layer chromatography (TLC), electrophoresis including capillaryelectrophoresis, affinity separations including immunoaffinityseparations, extraction methods including ethyl acetate or methanolextraction, and the use of chaotropic agents or any combination of theabove or the like. In some embodiments, liquid chromatography and/orSPE, and/or protein precipitation may be used in combination.

Protein precipitation is one method of preparing a test sample,especially a biological sample, such as serum or plasma. Proteinpurification methods are well known in the art, for example, Polson etal., Journal of Chromatography B 2003, 785:263-275, describes proteinprecipitation techniques suitable for use in methods of the presentinvention. Protein precipitation may be used to remove most of theprotein from the sample leaving IGF-I and/or IGF-II proteins in thesupernatant. The samples may be centrifuged to separate the liquidsupernatant from the precipitated proteins; alternatively the samplesmay be filtered to remove precipitated proteins. The resultantsupernatant or filtrate may then be applied directly to massspectrometry analysis; or alternatively to solid phase extraction and/orliquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, the use of protein precipitation such as forexample, acid ethanol protein precipitation, may obviate the need forTFLC, SPE, or other on-line extraction prior to mass spectrometry orHPLC and mass spectrometry.

In preferred embodiments, liquid-liquid extraction methods (such as acidethanol extraction) are used to extract native intact IGF-I and/orIGF-II from a sample. In these embodiments, between 10 μl and 500 μl ofsample, such as between 25 μl and 250 μl, such as about 100 μl, is addedto a portion of extraction solvent. The quantity of extraction solventis commensurate with sample volume and may vary depending on theextraction solvent used, but is preferably between about 50 μl and 1000μl. The sample/solvent mixtures are mixed and centrifuged, and a portionof the supernatant or organic phase (depending on solvent used) is drawnoff for further analysis. Solvent may be removed from the drawn offportion, for example under a nitrogen flow, and the residuereconstituted in a different solvent from that used for theliquid-liquid extraction. At least a portion of the resulting solutionmay then be subjected to additional processing steps, such as SPE and/orLC, prior to mass spectrometry.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including HPLC, rely on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess and may select LC, including HPLC, instruments and columns thatare suitable for use with IGF-I and/or IGF-II. The chromatographiccolumn typically includes a medium (i.e., a packing material) tofacilitate separation of chemical moieties (i.e., fractionation). Themedium may include minute particles, or may include a monolithicmaterial with porous channels. A surface of the medium typicallyincludes a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bondedor a cyano bonded surface. Alkyl bonded surfaces may include C-4, C-8,C-12, or C-18 bonded alkyl groups. In preferred embodiments, the columnis a C-18 alkyl bonded column (such as a Phenomenex Onyx monolithic C-18column). The chromatographic column includes an inlet port for receivinga sample and an outlet port for discharging an effluent that includesthe fractionated sample. The sample may be supplied to the inlet portdirectly, or from a SPE column, such as an on-line SPE guard cartridgeor a TFLC column.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In some embodiments, HPLC is conducted with an alkyl bonded analyticalcolumn chromatographic system. In certain embodiments, a C-18 analyticalcolumn (e.g., Phenomenex Onyx Monolithic C18, or equivalent) is used. Incertain embodiments, HPLC and/or TFLC are performed using HPLC Grade0.2% formic acid in water as mobile phase A and 0.2% formic acid inacetonitrile as mobile phase B.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, TFLC may be used for purification of an IGF-Iprotein or fragment prior to mass spectrometry. In such embodiments,samples may be extracted using a TFLC column which captures the analyte,then eluted and chromatographed on a second TFLC column or on ananalytical HPLC column prior to ionization. For example, sampleextraction with a TFLC extraction column may be accomplished with alarge particle size (50 μm) packed column. Sample eluted off of thiscolumn may then be transferred to an HPLC analytical column for furtherpurification prior to mass spectrometry. Because the steps involved inthese chromatography procedures may be linked in an automated fashion,the requirement for operator involvement during the purification of theanalyte can be minimized. This feature may result in savings of time andcosts, and eliminate the opportunity for operator error.

In some embodiments, protein precipitation is accomplished with acidethanol extraction from serum, and the resulting solution is subjectedto SPE, preferably conducted on-line with a C-18 extraction column(e.g., a Phenomenex Onyx C-18 guard cartridge, or equivalent). Theeluent from the SPE column may then be applied to an analytical LCcolumn, such as a HPLC column in an on-line fashion, prior to massspectrometric analysis.

Detection and Quantitation by Mass Spectrometry

Mass spectrometry is performed using a mass spectrometer, which includesan ion source for ionizing a sample and creating charged molecules forfurther analysis. In various embodiments, an IGF-I and/or IGF-II proteinmay be ionized by any suitable method known to the skilled artisan. Forexample, ionization of the sample may be performed by electronionization, chemical ionization, electrospray ionization (ESI), photonionization, atmospheric pressure chemical ionization (APCI),photoionization, atmospheric pressure photoionization (APPI), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. The skilled artisan will understand that the choice ofionization method may be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc. Depending on the particular ionization method andconditions employed, IGF-I and IGF-II proteins may be ionized to anumber of different charge states. The ionization source may be selectedto minimize the dispersion of charge states generated. In someembodiments, ESI (optionally heated) is used as the ionization source,and the ionization conditions are optimized to minimize the disbursementof observed multiply charged IGF-I and/or IGF-II protein ions.

IGF-I and/or IGF-II proteins may be ionized in positive or negativemode. In preferred embodiments, one or more IGF-I and/or IGF-II proteinsare ionized in positive mode. In some embodiments, multiply chargedintact IGF-I ions are generated with m/z ratios within the ranges ofabout 850.8±2, 957.1±2, 1093.7±2, and 1275.8±2. In some embodiments,multiply charged intact IGF-II ions are generated with m/z ratios withinthe ranges of about 934.69±2, 1068.07±2, 1245.92±2, and 1494.89±2. Themajority of the generated multiply charged ions within these ranges mayfall within a narrower sub-range, such as the indicated m/z±1.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass to charge ratio (m/z). Various analyzersfor determining m/z include quadrupole analyzers, ion trap analyzers,and time-of-flight analyzers, and orbitrap analyzers. According tomethods of the present invention, high resolution/high accuracy massspectrometry is used for quantitative analysis of IGF-I and/or IGF-IIproteins. That is, mass spectrometry is conducted with a massspectrometer capable of exhibiting a resolving power (FWHM) of at least10,000, with accuracy of about 50 ppm or less for the ions of interest;preferably the mass spectrometer exhibits a resolving power (FWHM) of20,000 or better and accuracy of about 20 ppm or less; such as aresolving power (FWHM) of 25,000 or better and accuracy of about 5 ppmor less; such as a resolving power (FWHM) of 25,000 or better andaccuracy of about 3 ppm or less. Three exemplary mass spectrometerscapable of exhibiting the requisite level of performance for IGF-Iand/or IGF-II protein ions are those which include orbitrap massanalyzers, certain TOF mass analyzers, or Fourier transform ioncyclotron resonance mass analyzers.

Elements found in biological active molecules, such as carbon, oxygen,and nitrogen, naturally exist in a number of different isotopic forms.For example, most carbon is present as ¹²C, but approximately 1% of allnaturally occurring carbon is present as ¹³C. Thus, some fraction ofnaturally occurring carbon containing molecules will contain at leastone ¹³C atom. Inclusion of naturally occurring elemental isotopes inmolecules gives rise to multiple molecular isotopic forms. Thedifference in masses of molecular isotopic forms is at least 1 atomicmass unit (amu). This is because elemental isotopes differ by at leastone neutron (mass of one neutron≈1 amu). When molecular isotopic formsare ionized to multiply charged states, the mass distinction between theisotopic forms can become difficult to discern because massspectrometric detection is based on the mass to charge ratio (m/z). Forexample, two isotopic forms differing in mass by 1 amu that are bothionized to a 5+ state will exhibit differences in their m/z of only 0.2(difference of 1 amu/charge state of 5). High resolution/high accuracymass spectrometers are capable of discerning between isotopic forms ofhighly multiply charged ions (such as ions with charges of ±5, ±6, ±7,±8, ±9, or higher).

Due to naturally occurring elemental isotopes, multiple isotopic formstypically exist for every molecular ion (each of which may give rise toa separately detectable spectrometric peak if analyzed with a sensitiveenough mass spectrometric instrument). The m/z ratios and relativeabundances of multiple isotopic forms collectively comprise an isotopicsignature for a molecular ion. In some embodiments, the m/z ratios andrelative abundances for two or more molecular isotopic forms may beutilized to confirm the identity of a molecular ion under investigation.In some embodiments, the mass spectrometric peak from one or moreisotopic forms is used to quantitate a molecular ion. In some relatedembodiments, a single mass spectrometric peak from one isotopic form isused to quantitate a molecular ion. In other related embodiments, aplurality of isotopic peaks are used to quantitate a molecular ion. Inthese later embodiments, the plurality of isotopic peaks may be subjectto any appropriate mathematical treatment. Several mathematicaltreatments are known in the art and include, but are not limited tosumming the area under multiple peaks or averaging the response frommultiple peaks.

An exemplary spectrum demonstrating such multiple isotopic forms ofIGF-I ions within a m/z range of about 1091-1095.5 is seen in FIG. 4. Asseen in the exemplary spectrum, peaks from various isotopic forms areobserved at m/z of about 1091.9447, 1092.8031, 1092.9445, 1093.0881,1093.2308, 1093.3740, 1093.5167, 1093.6597, 1093.8028, 1093.9458,1094.0889, 1094.2319, 1094.3754, 1094.5185, 1094.6606, and 1095.3717.Note, however, that the precise masses observed for isotopic variants ofany ion may vary slightly (e.g., ±0.1) because of instrumental variance.

Another exemplary spectrum demonstrating such multiple isotopic forms ofIGF-II ions within a m/z range of about 1067.0-1069.5 is seen in FIG.12. As seen in the exemplary spectrum, peaks from various isotopic formsare observed at m/z of about 1067.36, 1067.51, 1067.65, 1067.80,1067.94, 1068.08, 1068.23, 1068.37, 1068.51, 1068.65, 1068.80, 1068.94,and 1068.08. Again, the precise masses observed for isotopic variants ofany ion may vary slightly because of instrumental variance.

In mass spectrometric techniques generally, ions may be detected usingseveral detection modes. For example, selected ions may be detected,i.e. using a selective ion monitoring mode (SIM), or alternatively, ionsmay be detected using a scanning mode. When operated in a scanning mode,the mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). Further, when usinginstruments capable of multiple mass spectrometric events, such ascertain ion trap or triple quadrupole instruments, mass transitionsresulting from collision induced dissociation or neutral loss may bemonitored, e.g., multiple reaction monitoring (MRM) or selected reactionmonitoring (SRM).

The results of an analyte assay, that is, a mass spectrum, may berelated to the amount of the analyte in the original sample by numerousmethods known in the art. For example, given that sampling and analysisparameters are carefully controlled, the relative abundance of a givenion may be compared to a table that converts that relative abundance toan absolute amount of the original molecule. Alternatively, internal orexternal standards may be run with the samples, and a standard curveconstructed based on ions generated from those standards. Using such astandard curve, the relative abundance of a given ion may be convertedinto an absolute amount of the original molecule. In certain preferredembodiments, one or more standards are used to generate a standard curvefor calculating the quantity of an IGF-I and/or IGF-II protein. Methodsof generating and using such standard curves are well known in the artand one of ordinary skill is capable of selecting an appropriateinternal standard. For example, in preferred embodiments isotopicallylabeled or unlabeled intact non-human IGF-I and/or IGF-II (e.g.,recombinant mouse IGF-I and/or IGF-II) or isotopically labeled intacthuman IGF-I and/or IGF-II may be used as a standard. Numerous othermethods for relating the amount of an ion to the amount of the originalmolecule will be well known to those of ordinary skill in the art.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(²H), ¹³C, and ¹⁵N. The isotopic label can be incorporated at one ormore positions in the molecule and one or more kinds of isotopic labelscan be used on the same isotopically labeled molecule.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In some embodiments, intact IGF-I and/or IGF-II in a sample are detectedand/or quantified using MS as follows. The samples are subjected toliquid chromatography, preferably HPLC; the flow of liquid solvent froma chromatographic column enters a heated nebulizer interface of an ESIionization source; and the solvent/analyte mixture is converted to vaporin the heated charged tubing of the interface. The analyte (e.g., intactIGF-I and/or IGF-II) contained in the solvent, is ionized by applying alarge voltage to the solvent/analyte mixture. As the analyte exits thecharged tubing of the interface, the solvent/analyte mixture nebulizesand the solvent evaporates, leaving analyte ions in various chargestates. Quantitative data is then collected for the intensity of one ormore of ions. The quantitative data for signal intensity for one or moreions is then collected and related to the quantity of intact IGF-Iand/or IGF-II in the sample.

For intact IGF-I, ions in various charge states may be observed with m/zwithin the ranges of about 850.8±2 (9+), 957.1±2 (8+), 1093.7±2 (7+),and 1275.8±2 (6+). In some embodiments, data from one or more IGF-I ionswith m/z within the range of about 1093.7±2 is collected and used forquantitation. Exemplary ions within this m/z range include IGF-I ionswith m/z of about 1091.9±0.1, 1092.8±0.1, 1092.9±0.1, 1093.1±0.1,1093.2±0.1, 1093.4±0.1, 1093.5±0.1, 1093.7±0.1, 1093.8±0.1, 1093.9±0.1,1094.1±0.1, 1094.2±0.1, 1094.4±0.1, 1094.5±0.1, 1094.7±0.1, and1095.4±0.1. This listing is not meant to be limiting. Numerous otherions may be suitable for use in the instant methods, as demonstrated inthe spectrum shown in FIG. 3 (which demonstrates detection of groups ofisotopic ions at m/z of about 828.0509±2, 850.7373±2, 871.8730±2,920.5544±2, 939.6930±2, 956.9532±2, 975.4576±2, 1034.8128±2,1051.6337±2, 1073.9335±2, 1093.6597±2, 1114.5219±2, 1207.9401±2,1226.5705±2, 1252.5871±2, and 1275.6019±2; note, however, that as above,the ions of individual isotopes within these ranges will predominantlyfall within the ranges of the indicated m/z±1. Also, at this level ofprecision, masses observed for any ion may vary slightly because ofinstrumental variance, e.g. ±0.1).

For intact IGF-II, ions various charge states may be observed with m/zwithin the ranges of about 934.69±2 (8+), 1068.07±2 (7+), 1245.92±2(6+), and 1494.89±2 (5+). In some embodiments, data from one or moreIGF-II ions with m/z within the range of about 1068.07±2 is collectedand used for quantitation. Exemplary ions within this m/z range includeIGF-II ions with m/z of about 1067.36±0.1, 1067.51±0.1, 1067.65±0.1,1067.80±0.1, 1067.94±0.1, 1068.08±0.1, 1068.23±0.1, 1068.37±0.1,1068.51±0.1, 1068.65±0.1, 1068.80±0.1, 1068.94±0.1, and 1069.08±0.1. Insome embodiments, the one or more IGF-II ions are selected from thegroup consisting of IGF-II ions with m/z of about 1067.94±0.1 and1068.08±0.1. This listing is not meant to be limiting and other ions maybe suitable for use in the instant methods.

In some embodiments, the use of a high resolution/high accuracy massspectrometer may allow for the signal intensity of a peak from a singleisotopic form of a single ion (such as the single IGF-I ion peak shownin FIG. 4 at m/z of about 1093.66, or the single IGF-II peak shown inFIG. 12 at m/z of about 1067.80) to be selected for data acquisition.Alternatively, quantitative data for signal intensity from one or moreisotopic forms of a single ion (such as one or more IGF-I or IGF-IIisotopic forms as demonstrated in FIGS. 4 and 12), or signal intensityacross a narrow m/z range (such as all IGF-I signal intensity for a m/zrange of about 1093.7±1, or all IGF-II signal intensity for a m/z rangeof about 1068.2±1), may be collected and related to the quantity ofintact IGF-I and/or IGF-II in the sample.

In some embodiments, quantitative data for signal intensity is collectedfor one or more IGF-I and/or IGF-II ions from at least two differentcharge states. The intensities of these ions may then be used forquantitative assessment of intact IGF-I and/or IGF-II in the sample. Forexample, IGF-I may be quantitated with signal intensity from one or moreIGF-I ions at the 8+ charge state (i.e., IGF-I ions within a m/z rangeof about 957.1±2) and one or more IGF-I ions at the 7+ charge state(i.e., IGF-I ions within a m/z range 1093.7±2). In embodiments wherequantitative data for signal intensity of two or more ions arecollected, the intensities may be combined by any mathematical methodknown in the art (such as summation, or averaging the area under thecurves) for quantitative assessment of intact IGF-I and/or IGF-II in thesample.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks aremeasured to determine the amount of an IGF-I and/or IGF-II protein orfragment. As described above, the relative abundance of a given ion maybe converted into an absolute amount of the original analyte usingcalibration standard curves based on peaks of one or more ions of aninternal molecular standard.

In some embodiments, IGF-I and IGF-II are quantitated simultaneously. Inthese embodiments, each IGF-I and IGF-II may each be quantitated by anyof the methods provided above.

In certain preferred embodiments, the lower limit of quantitation (LLOQ)for IGF-I is within the range of about 15.0 ng/mL to 200 ng/dL,inclusive; preferably within the range of about 15.0 ng/dL to 100 ng/mL,inclusive; preferably within the range of about 15.0 ng/mL to 50 ng/mL,inclusive; preferably within the range of about 15.0 ng/mL to 25 ng/mL,inclusive; preferably within the range of about 15.0 ng/mL to 15 ng/mL,inclusive; preferably within the range of about 15.0 ng/mL to 10 ng/mL,inclusive; preferably about 15.0 ng/mL.

In certain preferred embodiments, the lower limits of quantitation(LLOQ) for IGF-II is within the range of about 30.0 ng/mL to 200 ng/dL,inclusive; preferably within the range of about 30.0 ng/dL to 100 ng/mL,inclusive; preferably within the range of about 30.0 ng/mL to 50 ng/mL,inclusive; preferably within the range of about 30.0 ng/mL to 25 ng/mL,inclusive; preferably within the range of about 30.0 ng/mL to 15 ng/mL,inclusive; preferably within the range of about 30.0 ng/mL to 10 ng/mL,inclusive; preferably about 30.0 ng/mL.

In certain preferred embodiments, the limit of detection (LOD) for IGF-Iis within the range of about 4.9 ng/mL to 200 ng/mL, inclusive;preferably within the range of about 4.9 ng/mL to 100 ng/mL, inclusive;preferably within the range of about 4.9 ng/mL to 50 ng/mL, inclusive;preferably within the range of about 4.9 ng/mL to 25 ng/mL, inclusive;preferably within the range of about 4.9 ng/mL to 20 ng/mL, inclusive;preferably about 4.9 ng/mL.

In certain preferred embodiments, the limits of detection (LOD) forIGF-II is within the range of about 8.2 ng/mL to 200 ng/mL, inclusive;preferably within the range of about 8.2 ng/mL to 100 ng/mL, inclusive;preferably within the range of about 8.2 ng/mL to 50 ng/mL, inclusive;preferably within the range of about 8.2 ng/mL to 25 ng/mL, inclusive;preferably within the range of about 8.2 ng/mL to 20 ng/mL, inclusive;preferably about 8.2 ng/mL.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods. In particular,the following Examples demonstrate quantitation of IGF-I and IGF-IIproteins or fragments by mass spectrometry with the use of a particularan internal standard. The use of the indicated internal standard is notmeant to be limiting in any way. Any appropriate chemical species,easily determined by one in the art, may be used as an internalstandard.

EXAMPLES Example 1: Enrichment of IGF-I Proteins or Fragments

Intact IGF-I was extracted from serum samples using a combination ofsample preparation and subsequent on-line SPE. Acid ethanol extractionwas conducted as follows.

100 μL of each serum sample was treated with 400 μL of acid/ethanol(87.5% EtOH/12.5% 2M HCl) to form a precipitate. The mixture was subjectto centrifugation to obtain a supernatant and pellet. 400 μL ofsupernatant is then withdrawn and mixed with 60 μL 1.5M Tris base. Anyprecipitate that formed with the addition of the Tris base was filteredout and discarded. The filtrate was diluted with an on-line dilutionsystem with 5% formic acid in water to reduce the ethanol concentrationto sufficient levels that the IGF-I in solution would bind to anextraction column.

The diluted extracted samples were injected into a Cohesive LC systemfor on-line SPE and HPLC processing prior to mass spectrometricanalysis. On-line extraction and enrichment of IGF-I was accomplishedusing a Phenomenex Monolithic Onyx C18 Guard Cartridge (10×4.6 mm) as anon-line SPE column. Analytical separation was accomplished by HPLC witha Phenomenex Onyx Monolithic C18 column (50×2.0 mm).

Example 2: Detection and Quantitation of Intact IGF-I with HighResolution/High Accuracy Orbitrap MS

MS was performed using a Thermo Exactive MS system (Thermo ElectronCorporation). This system employs an orbitrap MS analyzer capable ofhigh resolution/high accuracy MS. The instrument exhibited resolvingpower of approximately 25,000 FWHM, and mass accuracy of approximately 1ppm while measuring intact IGF-I.

Ionization was conducted with an ESI source in positive ion mode.Species of multiply charged intact IGF-I ions were observed with m/z ofabout 851, 957, 1094, and 1275, corresponding to the [IGF-I+nH]^(n+)(n=9+, 8+, 7+, and 6+, respectively) ions. Full scan spectra and anenlarged portion of this spectra showing the isotopic signature of the1094 ion are found in FIGS. 3 and 4, respectively.

Data collected for single isotopic forms of the two strongest ions,corresponding to isotopic forms of ions with m/z of about 956.9532 and1093.6592, were summed and use to quantitatively assess the amount ofintact IGF-I in the samples. Linearity was observed in a calibrationcurve prepared from 31 fmol to 4000 fmol on column of intact IGF-I. Thiscurve is shown in FIG. 5. The goodness of fit (R²) value for the nativeintact IGF-I was 0.9984.

Example 3: Detection and Quantitation of Intact IGF-I with HighResolution/High Accuracy TOF MS

MS was also performed using an Agilent 6530 Accurate-Mass Q-TOF MSsystem (Agilent Technologies, Inc.). This system employs a highresolution/high accuracy TOF MS analyzer capable of high resolution/highaccuracy MS. The instrument exhibited resolving power of approximately25,000 FWHM, and mass accuracy of approximately 3 ppm while measuringintact IGF-I. The following software was used for these experiments:Agilent MassHunter Workstation Acquisition B.02.01; Agilent MassHunterQuantitative Software B.03.02; Agilent MassHunter Qualitative softwareB.02.00; and Cohesive Aria OS v.1.5.1.

Exemplary spectra generated from samples at 40 fmol and 650 fmol in 100μL blank serum across the range of m/z of about 1090 to 1098 are shownin FIGS. 6A and 7A, respectively. Extracted ion chromatograms (EICs)corresponding to the spectra shown in FIGS. 6A and 7A are presented inFIGS. 6B and 7B, respectively.

Data were collected for isotopic forms of IGF-I ions with m/z of about1093.7±2, and the amount of intact IGF-I in the samples wasqualitatively and quantitatively assessed. Qualitative assessment (i.e.,confirmation of the identity of IGF-I based on the isotopic signature)was conducted by comparison of the spectra observed across the m/z rangeof about 1092 to 1095.3 with a calculated spectra based on naturallyoccurring isotopic distribution. The observed and calculated spectra areshown in FIGS. 8A and 8B, respectively.

Quantitative assessment was conducted with data from a single isotopicform (corresponding to a theoretical m/z of about 1093.5209), and withsummed data from multiple isotopic forms. Data from the single isotopicform was used to generate a linear calibration curve from 20 fmol to2600 fmol intact IGF-I on column. This corresponds to observation oflinearity over sample concentrations of about 8.2 ng/mL to about 1054ng/mL (with a sample size of 100 μL). Data collected for intact IGF-Iand the internal standard is presented in Table 1, below. Thecalibration curve is shown in FIGS. 9A (over the full concentrationrange tested) and 9B (expanded view of the low end of the concentrationrange). The goodness of fit (R²) value for the intact IGF-I was 0.9996.

TABLE 1 Intact IGF-I and internal standard (intact recombinant mouseIGF-I) determination for calibration curve Intact IGF-I InternalStandard Actual Retention Measured Retention Sample Concentration TimeIon Concentration Time Ion Number (fmol on column) (min) Count (fmol oncolumn) (min) Count 1 20.3 0.318 28441 28.3 0.249 1246400 2 40.6 0.32344032 45.4 0.251 1040110 3 81.3 0.312 87351 90.9 0.247 926927 4 162.50.330 137378 147.5 0.256 864934 5 325 0.330 280516 340.1 0.255 741062 6650 0.327 525627 645.7 0.255 722716 7 1300 0.328 1142551 1264.0 0.259797380 8 2600 0.325 2415290 2617.7 0.257 811149

Example 4: Accuracy and Precision of Quantitation of Intact IGF-I withHigh Resolution/High Accuracy TOF MS

The intra-assay precision was generated from assaying 10 replicates fromeach of six QC pools (in-house QC pools and Bio-Rad Tumor MarkerControls). The three QC pools were prepared by spiking known amounts ofIGF-I into stripped serum at levels of 100 ng/mL, 400 ng/mL, and 741ng/mL. The coefficient of variation (CV) for 10 replicates of a samplewas used to evaluate the reproducibility of quantitation. Data fromthese analyses are presented in Table 2 (for in-house QC pools) andTable 3 (for Bio-Rad Tumor Marker Controls).

TABLE 2 Intact IGF-I Intra-Assay Variation using In-House QC Pools QC 1QC 2 QC 3 Replicate (100 ng/mL) (400 ng/mL) (741 ng/mL) 1 96.0 385.6746.7 2 105.5 407.9 735.5 3 109.5 403.5 774.2 4 98.5 396.7 779.7 5 108.1399.4 776.6 6 105.7 389.0 754.7 7 104.3 405.0 775.7 8 98.4 410.2 756.3 999.9 386.8 752.7 10 102.0 395.3 729.4 Mean 102.8 397.9 758.2 SD 4.518.81 17.89 % CV 4.4 2.2 2.4

TABLE 3 Intact IGF-I Intra-Assay Variation using Bio-Rad Tumor MarkerControls Level 1 Level 2 Level 3 (Lot 19851) (Lot 19852) (Lot 19853)Replicate (ng/mL) (ng/mL) (ng/mL) 1 57.8 245.8 444.2 2 55.5 243.2 431.73 55.0 248.5 435.4 4 57.5 248.7 451.1 5 56.1 245.6 452.7 6 58.6 248.3451.3 7 58.5 247.3 429.7 8 58.0 234.2 446.6 9 56.6 241.9 445.5 10 56.9244.4 442.7 Mean 57.1 244.8 443.1 SD 1.23 4.37 8.25 % CV 2.2 1.8 1.9

Statistics performed on the results of quantitation demonstrated thatthe reproducibility (CV) for the six QC pools ranged from 2.4% to 4.4%for spiked in-house QC pools and from 1.8% to 2.2% for Bio-Rad TumorMarker Controls.

The inter-assay variation is defined as the reproducibility ofmeasurements between assays. The same six QC pools as above wereevaluated over 5 days. Data from these analyses are presented in Table 4(for in-house QC pools) and Table 5 (for Bio-Rad Tumor Marker Controls).

TABLE 4 Intact IGF-I Inter-Assay Variation for In-House QC Samples day 1day 2 day 3 day 4 day 5 In-House QC Pool 1 (100 ng/mL) run 1 88.5 96.0108.3 109.2 108.5 run 2 101.5 105.5 94.2 104.0 99.7 run 3 98.8 109.5105.4 98.8 106.2 run 4 104.3 98.5 101.7 104.8 109.3 run 5 100.8 108.1111.9 111.9 94.7 run 6 102.7 105.7 110.1 106.0 103.9 run 7 106.3 104.3100.3 105.1 105.8 run 8 107.8 98.4 112.3 104.1 105.3 Mean 101.3 103.2105.5 105.4 104.2 SD 6.0 5.0 6.4 3.9 4.8 % CV 5.9 4.8 6.1 3.7 4.6Accuracy 101.3 103.2 105.5 105.4 104.2 In-House QC Pool 2 (400 ng/mL)run 1 389.9 385.6 442.4 427.5 388.3 run 2 398.1 407.9 441.3 422.8 394.1run 3 394.1 403.5 430.8 400.9 406.2 run 4 414.6 396.7 469.0 448.1 406.7run 5 413.3 399.4 473.4 404.4 401.8 run 6 405.8 389.0 405.7 431.0 411.7run 7 414.2 405.0 408.6 392.6 401.2 run 8 412.4 410.2 428.5 423.3 388.3Mean 405.3 399.6 437.5 418.8 399.8 SD 10.0 8.8 24.8 18.3 8.7 % CV 2.52.2 5.7 4.4 2.2 Accuracy 101.3 99.9 109.4 104.7 100.0 In-House QC Pool 3(741 ng/mL) run 1 755.3 746.7 826.6 752.2 734.2 run 2 789.0 735.5 790.3724.1 729.9 run 3 737.2 774.2 782.6 757.9 742.3 run 4 773.2 779.7 807.4757.5 745.5 run 5 778.4 776.6 763.5 720.2 747.0 run 6 762.6 754.7 799.5744.7 751.1 run 7 764.9 775.7 836.9 747.6 724.1 run 8 781.1 756.3 742.3750.2 733.7 Mean 767.7 762.4 793.6 744.3 738.5 SD 16.5 16.4 31.3 14.49.4 % CV 2.1 2.2 3.9 1.9 1.3 Accuracy 103.6 102.9 107.1 100.5 99.7

TABLE 5 Intact IGF-I Inter-Assay Variation for Bio-Rad Tumor MarkerControls day 1 day 2 day 3 day 4 day 5 Bio-Rad Tumor Marker Control 1run 1 57.8 54.7 57.9 57.5 56.6 run 2 55.5 55.7 59.9 58.2 55.1 run 3 55.055.5 60.5 59.3 57.5 run 4 57.5 53.8 56.9 55.3 55.4 run 5 56.1 53.2 59.158.0 57.8 run 6 58.6 55.4 58.2 56.6 55.4 run 7 58.5 54.9 61.3 56.9 55.4Mean 57.0 54.7 59.1 57.4 56.2 SD 1.4 0.9 1.6 1.3 1.1 % CV 2.5 1.7 2.72.2 2.0 Bio-Rad Tumor Marker Control 2 run 1 245.8 235.6 258.9 249.7250.5 run 2 243.2 242.0 253.8 251.4 239.3 run 3 248.5 243.2 251.5 239.4246.2 run 4 248.7 237.3 254.0 249.2 259.7 run 5 245.6 246.6 257.4 248.6258.3 run 6 248.3 231.5 257.4 241.0 253.2 run 7 247.3 235.6 263.7 240.6258.0 Mean 246.8 238.8 256.7 245.7 252.2 SD 2.01 5.28 4.02 5.14 7.44 %CV 0.82 2.21 1.57 2.09 2.95 Bio-Rad Tumor Marker Control 3 run 1 444.2424.0 443.8 464.2 472.3 run 2 431.7 448.1 445.1 457.2 448.7 run 3 435.4439.3 458.4 467.5 461.8 run 4 451.1 435.1 470.4 457.2 438.0 run 5 452.7435.8 454.1 451.5 452.5 run 6 451.3 428.7 441.9 447.8 454.4 run 7 429.7433.3 424.2 440.2 454.0 Mean 442.3 434.9 448.3 455.1 454.5 SD 9.91 7.6714.58 9.41 10.65 % CV 2.24 1.76 3.25 2.07 2.34

Results of these measurements demonstrated that the inter-assayvariation (% CV) for the pools ranged from 1.3% to 6.1% for spikedin-house QC pools and from 0.8% to 3.3% for Bio-Rad Tumor MarkerControls. The overall variation for the low, medium, and high spikedin-house QC samples was 5.0%, 5.2%, and 3.5%, respectively, while theoverall variation for the low, medium, and high Bio-Rad QC material is3.3%, 3.1%, and 2.8%, respectively.

The intra-assay accuracy is defined as the accuracy of measurementswithin a single assay. Each in-house QC pool was assayed in 10replicates to determine the accuracy of repeatedly measuring intactIGF-I. The results for the QC pools yielded an accuracy of about 103%,99%, and 102% for the three pools, respectively.

The inter-assay accuracy is defined as the accuracy of measurementbetween assays. The three QC pools were analyzed in seven replicatesover five assays on four days to determine the accuracy of measuringintact IGF-I. The results for the analysis of the QC pools yielded anover all accuracy of 104%, 103%, and 103% for the three pools,respectively. These results are within the acceptable accuracy range of80% to 120%.

Example 4: Analyte Measurement Range for Quantitation of Intact IGF-Iwith High Resolution/High Accuracy TOF MS

Nine stripped serum samples were prepared spiked with intact IGF-Iacross a concentration range of 15 ng/mL to 2000 ng/mL. These sampleswere then analyzed on five separate days to assess analyte detectionrange and linearity of detection. A weighted linear regression from fiveconsecutive analyses yielded coefficient correlations of 0.995 orgreater, with an accuracy of ±20%. Thus, the quantifiable range of theassay is at least 15 ng/mL to 2000 ng/mL. A plot of the resultsdemonstrating linearity of response is shown in FIG. 10.

Example 5: Limit of Detection/Lower Limit of Quantitation of IntactIGF-I with High Resolution/High Accuracy TOF MS

The limit of detection (LOD) is the point at which a measured value islarger than the uncertainty associated with it and is definedarbitrarily as four standard deviations (SD) from the zeroconcentration. A blank was measured 22 times and the resulting arearatios were back calculated to establish a LOD of 4.9 ng/mL for intactIGF-I.

The lower limit of quantitation (LLOQ) is the point at which a measuredvalue is quantifiably meaningful. The analyte response at the LLOQ isidentifiable, discrete and reproducible with a precision of better thanor equal to 20% and an accuracy of between 80% and 120%. The LLOQ wasdetermined by assaying five different samples at concentrations close tothe expected LLOQ (4.9 ng/mL, 7.8 ng/mL, 15.6 ng/mL, 31.2 ng/mL, and62.5 ng/mL) and evaluating the intra-assay reproducibility in seven runsover five days. These analyses demonstrated that the LLOQ was 15 ng/mLfor intact IGF-I.

Example 6: Spike Recovery of Intact IGF-I with High Resolution/HighAccuracy TOF MS

A recovery study was performed by spiking patient serum with a known lowlevel of intact IGF-I with additional intact IGF-I to achieve finalconcentrations of 50 ng/mL, 100 ng/mL, 400 ng/mL, and 1000 ng/mL. Thespiked samples were analyzed, and the results corrected for backgroundlevels of intact IGF-I. Recoveries were calculated for each spikedconcentration, with mean recoveries being about 100%, 96%, 97%, and 92%,respectively. Data from these studies are shown in Table 6.

TABLE 6 Spike recovery studies for intact IGF-I in patient serum SpikeAmount 50 ng/mL 100 ng/mL 400 ng/mL 1000 ng/mL sample % recovery %recovery % recovery % recovery 1 92.6 95.2 96.4 97.7 2 88.3 102.3 96.789.9 3 118 91.0 98.2 89.0 average 99.8 96.1 97.1 92.2

Example 7: Inter-Method Correlation for Quantitation of Intact IGF-I

Samples from 100 patients were split and analyzed with the LC-MS methoddescribed above. Portions of the samples were also assayed using theSiemens IMMULITE 2000 immunoassay system (Siemens HealthcareDiagnostics, Inc.), the Meso Scale Discovery SECTOR system (Meso ScaleDiscovery), and RIA methods (conducted by Esoterix, Inc., Test Code500282, Blocking RIA after acid:alcohol extraction). Of the 100 splitsamples, 60 were analyzed with the IMMULITE system.

Data from the four methods were analyzed by Deming regression. Resultsof the comparisons are shown in Table 7. The LC-MS analysis wasdemonstrated to have the best agreement with the RIA method.

TABLE 7 Deming regression analysis of comparison of four intact IGF-Iassay methods Methods Variable Compared n m b Sy · x LC-MS vs RIA 100 1.039 ± 0.01572 −11.55 ± 5.673  36.1 LC-MS vs 60 0.8320 ± 0.02363 43.78± 11.49 63.1 IMMULITE LC-MS vs MSD 100 0.8310 ± 0.03174 46.12 ± 12.6086.6

Example 8: Intact IGF-I Interference Studies

The effects of hemolysis on intact IGF-I determination were evaluated bytitrating lysed red blood cells into patient serum to establishestimated hemoglobin concentrations of 0 mg/mL, 2.5 mg/mL, 5 mg/mL, 7.5mg/mL, 10 mg/mL, and 20 mg/mL. Three different patient samples weretitrated as described and extracted for intact IGF-I analysis. Theresults were compared to the non-spiked pool results and the percentdifference was calculated. Data generated for this comparison ispresented in Table 8.

TABLE 8 Hemolytic interference studies for intact IGF-I in patient serumsamples % Recovery Compared to Control Hemoglobin Concentration (mg/mL)0 2.5 5 7.5 10 20 sample 1 100.0 100.7 99.0 105.4 101.0 103.7 sample 2100.0 100.5 99.8 104.7 100.5 98.3 sample 3 100.0 98.0 105.1 110.7 104.1107.1

As seen in Table 8, all whole blood spiked samples yielded acceptableresults (80%-120% of control value) and demonstrated no dependencebetween intact IGF-I detection and hemoglobin concentration. Therefore,samples showing light to moderate hemolysis are acceptable.

The effects of lipemia on intact IGF-I determination were evaluated bytitrating brain lipid extract into patient serum to establish estimatedlipid concentrations of 0 mg/mL, 2.5 mg/mL, 5 mg/mL, 7.5 mg/mL, 10mg/mL, and 20 mg/mL. Three different patient samples were titrated asdescribed and extracted for IGF-I analysis. The results were compared tothe non-spiked pool results and the percent difference was calculated.Data generated for this comparison is presented in Table 9.

TABLE 9 Lipemic interference studies for intact IGF-I in patient serumsamples % Recovery Compared to Control Lipid Concentration (mg/mL) 0 2.55 7.5 10 20 sample 1 100.0 109.3 107.6 113.1 104.1 106.6 sample 2 100.0105.9 93.7 91.2 105.9 96.2 sample 3 100.0 103.3 102.6 101.7 94.4 112.6

As seen in Table 9, all lipid spiked samples yielded acceptable results(80%-120% of control value) and demonstrated no dependence betweenintact IGF-I detection and lipid concentration. Therefore, samplesshowing light to moderate lipemia are acceptable.

The effects of bilirubin on intact IGF-I determination were evaluated bytitrating bilirubin into patient serum to establish estimated bilirubinconcentrations of 0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL,and 2 mg/mL. Three different patient samples were titrated as describedand extracted for intact IGF-I analysis. The results were compared tothe non-spiked pool results and the percent difference was calculated.Data generated for this comparison is presented in Table 10.

TABLE 10 Bilirubin interference studies for intact IGF-I in patientserum samples % Recovery Compared to Control Bilirubin Concentration(mg/mL) 0 0.25 0.5 0.75 1 2 sample 1 100.0 84.3 91.4 97.9 89.3 94.3sample 2 100.0 87.5 96.5 88.5 90.5 104.0 sample 3 100.0 90.9 90.6 94.197.6 98.6

As seen in Table 10, all bilirubin spiked samples yielded acceptableresults (80%-120% of control value) and demonstrated no dependencebetween intact IGF-I detection and bilirubin concentration. Therefore,samples showing light to moderate bilirubin are acceptable.

The effects of IGFBP-3 on intact IGF-I determination were evaluated bytitrating recombinant IGFBP-3 into patient serum to establish estimatedIGFBP-3 concentrations of 0 mg/L, 2 mg/L, 5 mg/L, 8 mg/L, and 9 mg/L.Three different patient samples were titrated as described and extractedfor intact IGF-I analysis within three hours of their preparation.Another set of 15 patient samples were spiked with IGFBP-3 to a finalconcentration of 5 mg/L and equilibrated for three days at 4° C. beforeextraction. In both experiments, the results were compared to thenon-spiked pool results and the percent difference was calculated. Datagenerated for these comparisons are presented in Tables 11 and 12,respectively.

TABLE 11 IGFBP-3 interference studies for intact IGF-I in patient serumsamples (extracted 3 hours after preparation) % Recovery Compared toControl IGFBP-3 Concentration (mg/L) 2 5 8 9 Sample 1 80 94 92 106Sample 2 111 102 104 101 Sample 3 99 109 119 98

TABLE 12 IGFBP-3 interference studies for intact IGF-I in patient serumsamples (extracted 3 days after preparation) % Recovery Compared toSample Control (5 mg/L IGFBP-3) 1 96.7 2 111.7 3 117.0 4 116.2 5 105.9 6115.2 7 100.6 8 102.3 9 104.6 10 113.4 11 99.9 12 109.8 13 94.2 14 109.715 90.4 Mean 105%

As seen in Tables 11 and 12, all IGFBP-3 spiked samples yieldedacceptable results (80%-120% of control value) and demonstrated nodependence between intact IGF-I detection and IGFBP-3 concentration.Therefore, IGFBP-3 does not appear to interfere with the analysis ofIGF-I.

Example 9: IGF-I Sample Type Studies

Ten patient pools were collected in four Vacutainer® types: serum,citrate plasma, heparin plasma, and EDTA plasma. Levels of intact IGF-Iwere determined in samples from each sample type. A pairwise analysis ofvariance (ANOVA) only indicated statistically significant differencesbetween serum and citrate plasma. This indicates that serum, heparinplasma, and EDTA plasma are acceptable sample types. Data from thesestudies are found in Table 13.

TABLE 13 Effect of Sample Type on IGF-I Quantitation Intact IGF-I(measured value) EDTA Heparin Citrate Patient Serum plasma Plasma Plasma1 231.3 215.9 234.1 189.1 2 244.9 216.0 219.3 209.8 3 451.9 470.5 486.0376.6 4 289.4 252.3 264.3 232.7 5 545.4 491.3 540.4 465.9 6 213.1 208.7200.5 179.7 7 344.5 315.7 337.7 288.5 8 168.7 176.5 172.3 147.9 9 125.4112.3 116.2 108.3 10 217.9 210.7 209.1 173.6 p value n/a p > 0.05 p >0.05 p < 0.001 (compared to serum)

Example 10: Enrichment of IGF-II Proteins or Fragments

Intact human IGF-II was extracted from calibration, QC, and patientserum samples using a combination of off-line sample preparation andsubsequent on-line SPE and HPLC. Acid ethanol extraction was conductedas follows.

100 μL of each sample was treated with 400 μL of acid/ethanol (87.5%Etoh/12.5% 2M HCl) to form a precipitate. The mixture was subject tocentrifugation to obtain a supernatant and pellet. 350 μL of supernatantwas then withdrawn, mixed with 60 μL 1.5M Tris base, and incubated at−20° C. for 1 hour. The incubated mixture was then subjected tocentrifugation and any precipitate that formed with the addition of theTris base was discarded. The supernatant is then applied directly ontoan HPLC column for mass spectrometric analysis.

After the above sample preparation, the resulting solutions wereinjected into a Cohesive LC system for on-line SPE and HPLC processingprior to mass spectrometric analysis. On-line extraction and enrichmentof intact human IGF-II was accomplished using a Phenomenex MonolithicOnyx C18 Guard Cartridge (10×4.6 mm) as an on-line SPE column.Analytical separation was accomplished by HPLC with a Phenomenex OnyxMonolithic C18 column (50×2.0 mm).

Example 11: Detection and Quantitation of Intact IGF-II with HighResolution/High Accuracy TOF MS

MS was performed using an Agilent 6530 Accurate-Mass Q-TOF MS system(Agilent Technologies, Inc.). This system employs a high resolution/highaccuracy TOF MS analyzer capable of high resolution/high accuracy MS.The instrument exhibited resolving power of approximately 21,000 FWHM,and mass accuracy of approximately 3 ppm while measuring intact IGF-II.The following software was used for these experiments: AgilentMassHunter Workstation Acquisition B.02.01; Agilent MassHunterQuantitative Software B.03.02; Agilent MassHunter Qualitative softwareB.02.00; and Cohesive Aria OS v.1.5.1.

An exemplary spectrum generated from high resolution/high accuracy massspectrometric analysis of intact human IGF-II demonstrating intactIGF-II ions in charge states of 8+, 7+, 6+, and 5+ is shown in FIG. 11.This spectrum was collected across the range of m/z of about 400 to2000. As shown in FIG. 11, peaks from different charge states are seenat m/z of about 934.57, 1067.94, 1245.76, and 1494.71.

An exemplary spectrum generated from high resolution/high accuracy massspectrometric analysis of intact human IGF-II demonstrating intactIGF-II ions in a 7+ charge state is shown in FIG. 12. This spectrum wascollected across the range of m/z of about 1066 to 1070. As shown inFIG. 12, peaks from naturally occurring isotopes are seen at m/z ratiosof about 1067.36, 1067.51, 1067.65, 1067.80, 1067.94, 1068.08, 1068.23,1068.37, 1068.51, 1068.65, 1068.80, 1068.94, and 1068.08.

Data was collected for two isotopic forms of intact human IGF-II ionswith m/z of about 1067.94 and 1068.08, and the amount of intact humanIGF-II in the samples was qualitatively and quantitatively assessed.Qualitative assessment (i.e., confirmation of the identity of IGF-IIbased on the isotopic signature) was conducted by comparison of theexperimental isotopic ratio of the peaks at 106.94 and 1068.08 with atheoretical ratio calculated from naturally occurring isotopicdistribution.

Quantitative assessment was conducted with the sum of the two isotopicforms indicated above. A linear calibration curve was generated forcalibrator pool concentrations of about 15 ng/mL to about 2000 ng/mLintact human IGF-II. Data collected for intact human IGF-II is presentedin Table 14, below. The calibration curve is shown in FIG. 13 over thefull concentration range tested. The goodness of fit (R²) value for theintact human IGF-II was 0.9998.

TABLE 14 Intact human IGF-II determination for calibration curve Intacthuman IGF-II Actual Measured Sample Concentration Concentration AccuracyNumber (ng/mL) (ng/mL) (%) 1 7.81 6.5 82.9 2 15.63 17.3 110.9 3 31.2535.0 112.0 4 62.50 67.2 107.5 5 125.00 112.9 90.3 6 250.00 2335.9 94.4 7500.00 502.4 100.5 8 1000.00 1014.7 101.5 9 2000.00 2000.3 100.0

Example 12: Accuracy and Precision of Quantitation of Intact IGF-II withHigh Resolution/High Accuracy TOF MS

The intra-assay precision was generated from assaying 8 replicates fromeach of 6 QC pools (3 in-house QC pools and 3 off-the-clot human serumsamples). The coefficient of variation (CV) for 8 replicates of a samplewas used to evaluate the reproducibility of quantitation. Data fromthese analyses are presented in Table 15 (for in-house QC pools) andTable 16 (for off-the-clot human serum samples).

TABLE 15 Intact IGF-II Intra-Assay Variation using In-House QC Pools QC1 QC 2 QC 3 Replicate (200 ng/mL) (500 ng/mL) (1200 ng/mL) 1 193.6 497.01214.7 2 189.8 504.1 1205.9 3 197.4 504.1 1158.1 4 195.0 528.3 1197.2 5215.6 499.5 1205.3 6 200.5 498.9 1183.5 7 213.7 502.0 1231.7 8 205.3494.0 1242.9 Mean 201.4 503.5 1204.9 SD 9.4 10.6 26.6 % CV 4.7 2.1 2.2Accuracy 100.7 100.7 100.4

TABLE 16 Intact IGF-II Intra-Assay Variation using Off-the-Clot HumanSerum Samples Level 1 Level 2 Level 3 Replicate (ng/mL) (ng/mL) (ng/mL)1 43.7 224.6 446.6 2 44.9 237.9 441.1 3 36.9 224.4 452.4 4 38.9 233.6448.7 5 40.2 212.8 466.4 6 39.5 233.7 441.1 7 35.8 225.4 445.5 8 41.2220.2 460.4 Mean 40.1 226.6 450.3 SD 3.1 8.2 9.1 % CV 7.7 3.6 2.0

Statistics performed on the results of quantitation demonstrated thatthe reproducibility (CV) for the six QC pools ranged from 2.1% to 4.7%for spiked in-house QC pools and from 2.0% to 7.7% for off-the-clothuman serum samples.

The intra-assay accuracy is defined as the accuracy of measurementswithin a single assay. The repeated measurement of intact human IGF-IIin the in-house QC pools yielded accuracies of about 100.7%, 100.7%, and100.4% for pools at 200 ng/mL, 500 ng/mL, and 1200 ng/mL, respectively.

The inter-assay variation is defined as the reproducibility ofmeasurements between assays. The same 6 QC pools as above were evaluatedover 5 days. Data from these analyses are presented in Table 17 (forin-house QC pools) and Table 18 (for off-the-clot human serum samples).

TABLE 17 Intact IGF-II Inter-Assay Variation for In-House QC Samples.day 1 day 2 day 3 day 4 day 5 In-House QC Pool 1 (200 ng/mL) run 1 193.6208.7 196.7 235.3 196.1 run 2 189.8 216.4 210.8 224.9 199.4 run 3 197.4198.3 187.1 209.5 198.3 run 4 195.0 210.3 191.8 230.0 194.4 run 5 215.6197.3 196.8 200.9 198.1 run 6 200.5 223.5 191.2 — 201.8 run 7 213.7204.2 185.9 218.5 196.0 run 8 205.3 — 192.0 224.2 198.7 Mean 201.4 208.4194.1 220.5 197.9 SD 9.4 9.5 7.8 11.9 2.3 % CV 4.7 4.5 4.0 5.4 1.2Accuracy 100.7 104.2 97.0 110.2 98.9 In-House QC Pool 2 (500 ng/mL) run1 497.0 494.2 523.9 494.5 457.9 run 2 504.1 — 496.2 491.5 499.1 run 3504.1 498.9 514.5 502.9 471.7 run 4 528.3 482.3 468.5 491.5 500.2 run 5499.5 505.5 496.8 490.1 493.3 run 6 498.9 475.8 476.6 529.8 501.7 run 7502.0 464.7 506.9 492.9 486.4 run 8 494.0 489.3 513.8 484.9 507.0 Mean503.5 487.2 499.6 497.3 489.7 SD 10.6 14.1 19.2 14.1 16.9 % CV 2.1 2.93.8 2.8 3.5 Accuracy 100.7 97.4 99.9 99.5 97.9 In-House QC Pool 3 (1200ng/mL) run 1 1214.7 1155.8 1201.9 1287.5 1188.8 run 2 1205.9 1218.01116.1 1281.5 1262.6 run 3 1158.1 1141.0 1202.9 1104.4 1165.1 run 41197.2 1110.8 1106.8 1327.7 1168.8 run 5 1205.3 1055.3 1248.1 1265.91146.5 run 6 1183.5 1221.2 1184.0 1207.0 1250.2 run 7 1231.7 1233.41290.4 1158.0 1062.2 run 8 1242.9 1088.1 1191.7 1152.8 1151.9 Mean1204.9 1153.0 1192.7 1223.1 1174.5 SD 26.6 66.6 61.1 79.1 62.9 % CV 2.25.8 5.1 6.5 5.4 Accuracy 100.4 96.1 99.4 101.9 97.9

TABLE 18 Intact IGF-II Inter-Assay Variation for Off-the-Clot HumanSerum Samples. day 1 day 2 day 3 day 4 day 5 Off-the-Clot Human SerumSample 1 run 1 43.7 43.5 40.1 44.8 42.8 run 2 44.9 37.7 43.0 43.9 44.0run 3 36.9 43.1 43.7 44.7 43.5 run 4 38.9 40.2 39.6 42.5 40.9 run 5 40.242.9 40.8 42.9 42.4 run 6 39.5 38.2 41.5 41.6 43.6 run 7 35.8 36.4 38.342.3 41.7 run 8 41.2 34.3 37.7 39.7 42.9 Mean 40.1 39.5 40.6 42.8 42.7SD 3.1 3.4 2.1 1.7 1.0 % CV 7.7 8.7 5.2 3.9 2.4 Off-the-Clot Human SerumSample 2 run 1 224.6 222.4 228.8 216.3 228.2 run 2 237.9 227.8 222.9214.6 226.4 run 3 224.4 217.2 236.4 222.8 222.4 run 4 233.6 224.5 217.5214.4 235.4 run 5 212.8 222.1 244.2 226.4 219.2 run 6 233.7 229.7 223.8220.4 222.7 run 7 225.4 233.7 225.6 213.3 211.3 run 8 220.2 220.5 230.2210.9 235.7 Mean 226.6 224.7 228.7 217.4 225.2 SD 8.2 5.4 8.4 5.3 8.2 %CV 3.6 2.4 3.7 2.4 3.6 Off-the-Clot Human Serum Sample 3 run 1 446.6446.5 453.6 454.4 448.5 run 2 441.1 454.3 439.5 456.5 450.4 run 3 452.4469.6 453.8 450.2 442.3 run 4 448.7 452.9 453.3 456.8 434.3 run 5 466.4464.5 461.0 460.8 458.1 run 6 441.1 442.3 441.2 461.2 444.6 run 7 445.5454.5 443.4 450.5 457.9 run 8 460.4 469.4 441.1 430.2 450.6 Mean 450.3456.8 448.4 452.6 448.3 SD 9.1 10.2 8.0 9.9 8.0 % CV 2.0 2.2 1.8 2.2 1.8

Results of these measurements demonstrated that the inter-assayvariation (% CV) for the pools ranged from 3.2% to 6.1% for spikedin-house QC pools and from 2.0% to 6.5% for off-the-clot human serumsamples.

The inter-assay accuracy is defined as the accuracy of measurementbetween assays.

The repeated measurement of intact human IGF-II in the in-house QC poolsyielded accuracies of about 102.2%, 99.1%, and 99.1% for the pools at200 ng/mL, 500 ng/mL, and 1200 ng/mL, respectively. These results arewithin the acceptable accuracy range of 80% to 120%.

Example 13: Limit of Detection/Lower Limit of Quantitation of IntactIGF-II with High Resolution/High Accuracy TOF MS

The limit of blank (LOB) is the point at which a measured value islarger than the uncertainty associated with it and is definedarbitrarily as 2 standard deviations (SD) from the zero concentration.Blank samples of the appropriate biological matrix (stripped serum) wereobtained and measured 15 times. The resulting area ratios were backcalculated to establish a LOB of 4.9 ng/mL of intact human IGF-II instripped serum.

The limit of detection (LOD) is the point at which a measured value islarger than the uncertainty associated with it and is definedarbitrarily as four standard deviations (SD) from the zeroconcentration. A blank was measured 15 times and the resulting arearatios were back calculated to establish a LOD of 8.2 ng/mL for intacthuman IGF-II.

The lower limit of quantitation (LLOQ) is the point at which a measuredvalue is quantifiably meaningful. The analyte response at the LLOQ isidentifiable, discrete and reproducible with a precision of better thanor equal to 20% and an accuracy of between 80% and 120%. The LLOQ wasdetermined by assaying five different samples at concentrations close tothe expected LLOQ (4.9 ng/mL, 7.8 ng/mL, 15.6 ng/mL, 31.2 ng/mL, and62.5 ng/mL) and evaluating the intra-assay reproducibility in six runsover five days. These analyses demonstrated that the LLOQ was 30 ng/mLfor intact human IGF-II.

Example 14: Spike Recovery of Intact IGF-II with High Resolution/HighAccuracy TOF MS

A recovery study was performed by spiking stripped serum with intacthuman IGF-II to achieve final concentrations of 62.5 ng/mL, 125 ng/mL,500 ng/mL, and 1200 ng/mL. The spiked samples were analyzed, and theresults corrected for background levels of intact human IGF-II.Recoveries were calculated for each spiked concentration, with meanrecoveries being about 106%, 104%, 99%, and 99%, respectively. Data fromthese studies are shown in Table 19.

TABLE 19 Spike recovery studies for intact IGF-II in patient serum SpikeAmount 62.5 ng/mL 125 ng/mL 500 ng/mL 1200 ng/mL sample % recovery %recovery % recovery % recovery 1 99.1 101.1 100.7 100.4 2 101.9 101.497.4 96.1 3 116.7 109.5 99.9 99.4 average 105.9 104.0 99.3 98.6

Example 15: Inter-Method Correlation for Quantitation of Intact IGF-II

Samples from 42 patients were split and analyzed with the LC-MS methoddescribed above. Portions of the samples were also assayed using an IRMAmethodology performed by Quest Diagnostics Nichols Institute.

Data from the two methods were analyzed by linear and Deming regression.Results of the comparisons are shown in Table 20 and FIG. 13. The LC-MSanalysis was demonstrated to have good agreement with the IRMA method.

TABLE 20 Comparison of LC-MS and IRMA assay methods for intact IGF-IIIGF-II by LC-MS vs IRMA Variable Comparison n m b R² Linear regression42 0.9829 ± 0.07627 −28.10 ± 53.02 0.82 Deming regression 42  1.171 ±0.09090 −153.0 ± 63.19 n/a

Example 16: IGF-II Interference Studies

The effects of hemolysis on intact human IGF-II determination wereevaluated by titrating lysed red blood cells into patient serum toestablish estimated hemoglobin concentrations of 0 mg/mL, 2.5 mg/mL, 5mg/mL, 7.5 mg/mL, 10 mg/mL, and 20 mg/mL. Three different patientsamples were titrated as described and extracted for intact human IGF-IIanalysis. The results were compared to the non-spiked pool results andthe percent difference was calculated. Data generated for thiscomparison is presented in Table 21.

TABLE 21 Hemolytic interference studies for intact human IGF-II inpatient serum samples % Recovery Compared to Control HemoglobinConcentration (mg/mL) 0 2.5 5 7.5 10 20 sample 1 100.0 99.3 104.9 94.4102.1 92.4 sample 2 100.0 102.7 101.1 98.9 102.7 103.3 sample 3 100.095.8 102.7 100.6 103.0 109.5

As seen in Table 21, all whole blood spiked samples yielded acceptableresults (80%-120% of control value) and demonstrated no dependencebetween intact human IGF-II detection and hemoglobin concentration.Therefore, samples showing light to moderate hemolysis are acceptable.

The effects of lipemia on intact human IGF-II determination wereevaluated by titrating brain lipid extract into patient serum toestablish estimated lipid concentrations of 0 mg/mL, 2.5 mg/mL, 5 mg/mL,7.5 mg/mL, 10 mg/mL, and 20 mg/mL. Three different patient samples weretitrated as described and extracted for human IGF-II analysis. Theresults were compared to the non-spiked pool results and the percentdifference was calculated. Data generated for this comparison ispresented in Table 22.

TABLE 22 Lipemic interference studies for intact human IGF-II in patientserum samples % Recovery Compared to Control Lipid Concentration (mg/mL)0 2.5 5 7.5 10 20 sample 1 100.0 102.9 107.2 112.9 112.9 107.2 sample 2100.0 106.2 95.5 100.0 95.3 106.5 sample 3 100.0 108.0 93.0 95.1 92.0106.6

As seen in Table 22, all lipid spiked samples yielded acceptable results(80%-120% of control value) and demonstrated no dependence betweenintact human IGF-II detection and lipid concentration. Therefore,samples showing light to moderate lipemia are acceptable.

The effects of bilirubin on intact human IGF-II determination wereevaluated by titrating bilirubin into patient serum to establishestimated bilirubin concentrations of 0 mg/mL, 0.25 mg/mL, 0.5 mg/mL,0.75 mg/mL, 1 mg/mL, and 2 mg/mL. Three different patient samples weretitrated as described and extracted for intact human IGF-II analysis.The results were compared to the non-spiked pool results and the percentdifference was calculated. Data generated for this comparison ispresented in Table 23.

TABLE 23 Bilirubin interference studies for intact human IGF-II inpatient serum samples % Recovery Compared to Control BilirubinConcentration (mg/mL) 0 0.25 0.5 0.75 1 2 sample 1 100.0 87.0 92.1 100.996.8 108.3 sample 2 100.0 84.6 93.1 81.0 87.0 90.7 sample 3 100.0 115.3108.9 99.5 89.2 104.9

As seen in Table 23, all bilirubin spiked samples yielded acceptableresults (80%-120% of control value) and demonstrated no dependencebetween intact human IGF-II detection and bilirubin concentration.Therefore, samples showing light to moderate bilirubin are acceptable.

The effects of IGFBP-3 on intact human IGF-II determination wereevaluated by titrating recombinant IGFBP-3 into patient serum toestablish estimated IGFBP-3 concentrations of 5 mg/L, 10 mg/L, and 20mg/L. Three different patient samples were titrated as described andextracted for intact human IGF-II analysis within three hours of theirpreparation. Another set of 15 patient samples were spiked with IGFBP-3to a final concentration of 5 mg/L and equilibrated for three days at 4°C. before extraction. In both experiments, the results were compared tothe non-spiked pool results and the percent difference was calculated.Data generated for these comparisons are presented in Tables 24 and 25,respectively.

TABLE 24 IGFBP-3 interference studies for intact human IGF-II in patientserum samples (extracted 3 hours after preparation) % Recovery Comparedto Control IGFBP-3 Concentration (mg/L) 5 10 20 Sample1 104 108 107Sample 2 101 95 102 Sample 3 101 102 102

TABLE 25 IGFBP-3 interference studies for intact human IGF-II in patientserum samples (extracted 3 days after preparation) % Recovery Comparedto Sample Control (5 mg/L IGFBP-3) 1 102.6 2 111.0 3 117.5 4 105.6 591.9 6 87.5 7 111.2 8 105.9 9 99.1 10 111.8 11 104.6 12 95.5 13 99.9 1487.7 15 99.3 Mean 102%

As seen in Tables 24 and 25, all IGFBP-3 spiked samples yieldedacceptable results (80%-120% of control value) and demonstrated nodependence between intact human IGF-II detection and IGFBP-3concentration. Therefore, IGFBP-3 does not appear to interfere with theanalysis of intact human IGF-II.

Example 17: IGF-II Sample Type Studies

Ten patient pools were collected in four Vacutainer® types: serum,citrate plasma, heparin plasma, and EDTA plasma. Levels of intact humanIGF-II were determined in samples from each sample type. A pairwiseanalysis of variance (ANOVA) only indicated statistically significantdifferences between serum and citrate plasma. This indicates that serum,heparin plasma, and EDTA plasma are acceptable sample types. Data fromthese studies are found in Table 26.

TABLE 26 Effect of Sample Type on IGF-II Quantitation Intact HumanIGF-II (measured value) EDTA Heparin Citrate Patient Serum plasma PlasmaPlasma 1 497.9 469.5 490.2 432.4 2 790.1 783.0 726.0 726.0 3 593.3 584.1681.3 650.0 4 791.7 723.8 714.4 591.3 5 631.7 603.4 754.8 623.7 6 756.0708.1 744.2 717.5 7 642.7 598.7 648.2 612.1 8 652.9 606.1 658.2 599.3 9585.4 557.0 551.0 523.3 10 840.0 875.2 862.1 721.8 p value n/a p > 0.05p > 0.05 p < 0.05 (compared to serum)

Example 18: Simultaneous Quantitation of IGF-II and IGF-I

Samples from 12 patients were prepared and analyzed with the LC-MSmethod described above. Intact human IGF-I and IGF-II were quantitatedsimultaneously for each sample. The results of the simultaneous analysisare presented in Table 27.

TABLE 27 Simultaneous Quantitation of IGF-II and IGF-I Patient IGF-II(ng/mL) IGF-I (ng/mL) 1 316 371 2 207 1107 3 389 416 4 495 279 5 297 8366 408 353 7 301 45 8 339 227 9 83 35 10 339 214 11 291 731 12 477 216 6408 353

An exemplary Total Ion Chromatogram and Extracted Ion Chromatograms fromIGF-II and IGF-I from these studies are shown in FIGS. 15A, 15B, and15C, respectively.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount ofinsulin-like growth factor-I (IGF-I) in a sample, the method comprising:a. ionizing IGF-I in the sample to produce one or more ions detectableby mass spectrometry; b. determining the amount of one or more of theions comprising an ion with a mass-to-charge ratio selected from thegroup consisting of 850.8±2 and 957.1±2 by mass spectrometry; and c.determining the amount of the IGF-I in the sample using the amount ofthe determined ion or ions.
 2. The method of claim 1, wherein the methoddoes not include digesting the protein prior to ionization.
 3. Themethod of claim 1, wherein the mass spectrometry is conducted with amass analyzer capable of a full width at half maximum (FWHM) of greaterthan or equal to 10,000 and an accuracy of less than or equal to 50 ppm.4. The method of claim 1, wherein the method comprises determining theamount of an intact long R3 IGF-I.
 5. The method of claim 1, furthercomprising chemically modifying the protein, prior to ionization, toreduce the number of disulfide bridges in the protein.
 6. The method ofclaim 1, further comprising purifying the protein with solid phaseextraction (SPE) prior to ionization.
 7. The method of claim 1, furthercomprising purifying the protein by high performance liquidchromatography (HPLC) prior to ionization.
 8. The method of claim 1,wherein with the mass analyzer is a time of flight mass analyzer capableof a FWHM of greater than or equal to 20,000 and an accuracy of lessthan or equal to 10 ppm.
 9. The method of claim 1, wherein the samplecomprises plasma or serum.
 10. The method of claim 1, wherein the one ormore ions used to determine the amount of the IGF-I protein furthercomprise one or more ions selected from the group of ions with a mass tocharge ratio of 1091.9447±0.1, 1092.8031±0.1, 1092.9445±0.1,1093.0881±0.1, 1093.2308±0.1, 1093.3740±0.1, 1093.5167±0.1,1093.6597±0.1, 1093.8028±0.1, 1093.9458±0.1, 1094.0889±0.1,1094.2319±0.1, 1094.3754±0.1, 1094.5185±0.1, 1094.6606±0.1, and1095.3717±0.1.